Liver cancer methylation markers and uses thereof
a methylation marker and liver cancer technology, applied in the field of liver cancer methylation markers, can solve the problems of high cancer morbidity and mortality, and high cost of invasive and sometimes time-consuming procedures
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example 1
ethodology
[0193]Tumor DNA Extraction
[0194]Genomic DNA extraction from pieces of freshly frozen healthy or cancer tissues was performed with QIAamp DNA Mini Kit (Qiagen) according to manufacturer's recommendations. DNA was extracted from roughly 0.5 mg of tissue. DNA was stored at −20° C. and analyzed within one week of preparation.
[0195]DNA Extraction from FFPE Samples
[0196]Genomic DNA from frozen FFPE samples was extracted using QIAamp DNA FFPE Tissue Kit with several modifications. DNA were stored at −20° C. for further analysis.
[0197]Bisulfite Conversion of Genomic DNA
[0198]1 μg of genomic DNA was converted to bis-DNA using EZ DNA Methylation-Lightning™ Kit (Zymo Research) according to the manufacturer's protocol. Resulting bis-DNA had a size distribution of ˜200-3000 bp, with a peak around ˜500-1000 bp. The efficiency of bisulfite conversion was >99.8% as verified by deep-sequencing of bis-DNA and analyzing the ratio of C to T conversion of CH (non-CG) dinucleotides.
[0199]Determ...
example 2
cer Diagnostic
[0208]Patient data was obtained from the Cancer Genome Atlas (TCGA). DNA methylation data were obtained from the TCGA analysis of about 450,000 sites generated using the Infinium 450K Methylation Array. Methylation profiles for liver cancer tissue and normal liver tissue were analyzed. Four clinical covariates were used, which includes:
[0209]Age (continuous);
[0210]Gender (categorical) with two levels: Female / Male;
[0211]Race (categorical) with three levels: Asian, Black or African American and White. The category American Indian or Alaska Native was removed due to insufficient number of observations;
[0212]American Join Committee on Cancer (AJCC) stage—combined into a four-level covariate: Stage I, Stage II, Stage III and Stage IV.
[0213]The data were further modified by removal of missing value in any of the four clinical and demographic covariates to generate 377 liver cancer samples and 50 normal liver tissue samples for subsequent diagnostic analysis.
[0214]Six additio...
example 3
Analysis
[0227]DNA methylation data were obtained from the TCGA analysis of about 485,000 sites generated using the Infinium 450K Methylation Array. Methylation profile for liver cancer tissue was analyzed. Four clinical covariates were used as described in Example 1.
[0228]The data were further modified by removal of 1) negative and 0 survival time; or 2) missing value in any of the four clinical and demographic covariates; to generate 355 liver cancer samples with 98 events (the event of interest was defined as death) for subsequent diagnostic analysis.
[0229]Additional datasets were also obtained from TCGA which includes:
[0230]Colon cancer: 365 observations with 53 events;
[0231]Breast cancer: 967 observations with 103 events;
[0232]Lung adenocarcinoma (LUAD): 516 observations with 141 events;
[0233]Lung squamous cell carcinoma (LUSC): 418 observations with 150 events;
[0234]and were used during the subsequent diagnostic analysis.
[0235]For each type of cancer: breast, colon, liver, and ...
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